CN104177460B - The preparation method of a kind of 3,5-disaccharides anthocyanin - Google Patents
The preparation method of a kind of 3,5-disaccharides anthocyanin Download PDFInfo
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Abstract
The present invention relates to natural colouring matter preparing technical field, relate in particular to the preparation method of a kind of 3,5-disaccharides anthocyanin. The method comprises the step of ultrasonic assisted extraction, extraction, purifying. The method with ultrasonic assisted extraction reduced extractant use, shorten extraction time, improved extraction efficiency. The method that adopts gel column to combine with macroreticular resin, removes impurity, separates anthocyanin monomer, thereby has avoided the use of preparation or half preparative high-performance liquid chromatographic, has simplified operation; And gel column and macroreticular resin filler can Reusabilities, can economize on resources. Experimental results show that: method provided by the invention is extracted 3,5-disaccharides anthocyanin, and every g mountain Grape Skin dry powder can obtain 3 of 7.36mg, 5-disaccharides anthocyanin, extraction efficiency and purity are all higher.
Description
Technical field
The present invention relates to natural colouring matter preparing technical field, relate in particular to the system of a kind of 3,5-disaccharides anthocyaninPreparation Method.
Background technology
In recent years, day by day serious along with food and drug safety problem, the exploitation of natural colouring matter and application oneselfBecome the problem of every profession and trade scientific worker common concern. In recent years, natural colouring matter in the international marketGrowth rate all remains on more than 10% always. Natural colouring matter mostly derive from natural plants root, stem, leaf,Flower, fruit etc., therefore, adopt these natural colouring matters safer, also more can obtain consumer's favor.Anthocyanin class pigment is as a large class of natural colouring matter, because it enriches gorgeous color and luster and antioxidation activity,Be widely used in food, medicine and other fields at present.
Anthocyanin (anthocyanin) is a class hydroxyl and methylated 2-phenyl-chromene melt ion (flowerPigment, anthocyanidin) polyphenol compound that is combined into by glycosidic bond with glycan molecule. AnthocyanidinBelonging to flavonoids, is a kind of aldehydes matter, because of benzene ring substitution group, hydroxyl and methoxyl group quantity and positionDifference, anthocyanidin monomer mainly comprises following six classes: delphinidin, methyl delphinidin, dimethyl delphinidin,Anthocyanidin, peonidin, pelargonidin. In these anthocyanidin monomers, delphinidin is least stable, diformazanDelphinidin is the most stable. And main anthocyanin can be divided into according to the difference that becomes glycosides glycosyl position and quantityFour classes, are respectively 3-monosaccharide anthocyanin, 3-disaccharide class anthocyanin, 3,5-disaccharides anthocyanin and 3,7-disaccharidesClass anthocyanin etc. In the anthocyanin of this Four types, disaccharide glycoside pigment is because containing disaccharide molecule in molecule,Its stability is apparently higher than corresponding monosaccharide material. Therefore, disaccharide anthocyanin is as natural colouring matter,In food and medicine industry, there is very high researching value and application prospect. But, owing to lacking cheaplyPreparation technology causes at China's anthocyanin class little as the production of natural food colour, and price is very highExpensive, be difficult to be applied to suitability for industrialized production.
Anthocyanin is extensively present in purple sweet potato, grape, blood orange, red cabbage, blueberry, eggplant skin, cherryThe group of the plants such as peach, blood orange, the red certain kind of berries, strawberry, mulberries, hawthorn skin, purple perilla, black (red) rice, morning gloryIn knitting. Wherein with the content in grape compared with horn of plenty. Result of study in recent years shows, vitis vinifera(VitisamurensisL.) in, only contain monosaccharide anthocyanin, and mountain grape (VitisamurensisRupr.)The content of middle disaccharide anthocyanin is very abundant. And mountain grape is wide at China's distribution area, utilize mountain grapeHave a good application prospect as material extraction disaccharide class anthocyanin. Although the extraction to anthocyanin at presentThe existing report in many ways of method, still, the extraction that these existing methods is applied to disaccharide anthocyanin is pastToward there is the problems such as recovery rate is low, purity is not enough, therefore, the extraction purifying of probing into disaccharide class anthocyanin dividesSignificant from method.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide the system of a kind of 3,5-disaccharides anthocyaninPreparation Method. The method recovery rate is high, and gained 3.5-disaccharides anthocyanin purity is higher; And without by preparationType or Semipreparative chromatography, simple to operate; The method agents useful for same toxicity is lower, to environmentFriendly.
Provided by the invention 3, the preparation method of 5-disaccharides anthocyanin, comprises the following steps:
Step 1: with the ultrasonic extraction of ethanolic solution mountain Grape Skin powder, extract is removed ethanol, makes first and carriesGet liquid;
Step 2: extract the first extract with ethyl acetate, water intaking layer, removal ethyl acetate makes second and carriesGet liquid;
Step 3: the second extract is splined on to XAD-7 macroporous resin column, after distilled water flushing, withEthanolic solution wash-out, obtains the first eluent, and drying, makes crude extract;
Step 4: crude extract, with dissolve with methanol solution, is splined on to SephadexLH-20 gel after filtrationPost, with methanol solution wash-out, obtains the second eluent, and drying, to obtain final product;
Wherein, in ethanolic solution, the volume fraction of each component is: ethanol 60%, formic acid 2%, water 38%;
In methanol solution, the volume fraction of each component is: methyl alcohol 20%, formic acid 2%, water 78%;
The mass volume ratio of mountain Grape Skin powder and ethanolic solution is: 1g:10mL;
The temperature of ultrasonic extraction is 53.5 DEG C, and the time is 20min, and number of times is 3 times.
Method provided by the invention, with in ultrasonic assisted extraction Grape Skin anthocyanin, utilize ultrasonic waveIn solution, form cavitation effect, make more multi-solvent be penetrated into sample substrate, increase connecing between solid liquid phaseTouch area, solute is dissolved into solvent as soon as possible, thus reduced extractant use, shorten extract timeBetween, improved extraction efficiency.
In the first extract of ultrasonic extraction, still contain sugar, albumen and other non-anthocyanin aldehydes matters,The present invention, by the method that adopts gel column to combine with macroreticular resin, removes impurity, separates anthocyanin listBody, thus the use of preparation or half preparative high-performance liquid chromatographic avoided, simplify operation; And, solidifyingGlue post and macroreticular resin filler can Reusabilities, can economize on resources.
The preparation method of mountain of the present invention Grape Skin powder is: get hill grape and peel off pericarp in fact, reallySkin, through liquid nitrogen flash freezer, refiner abrasive dust, freeze dryer 24h freeze drying, to obtain final product.
The present invention is by the experiment proved that:
In ethanolic solution, the volume fraction of alcohol can affect extraction effect, and the recovery rate of anthocyanin is along with alcohol volumeThe increase of mark, anthocyanin extracted amount increases, but in the time that alcohol volume fraction is after 60%, anthocyaninContent increasing degree tends towards stability;
The mass volume ratio of mountain Grape Skin powder and ethanolic solution can affect extraction effect: recovery rate is with liquid ratioIncrease and increase, in the time being increased to 10:1 rise no longer increase.
The temperature of ultrasonic extraction impacts extraction effect: for the impact of extracting temperature, through response surfaceAnalysis result shows, extraction efficiency reaches peak in the time of 53.5 DEG C.
The time of ultrasonic extraction impacts extraction effect: in 20min, extend and carry with extraction timeTaken amount increases gradually, and after this extracted amount declines slightly, may be because long-time high temperature extracts, and causes flowerLook glycosides decomposes.
Extraction time impacts extraction effect: along with the increase of extraction time, anthocyanin recovery rate increasesAdd, while extraction for the third time, anthocyanin extraction rate reached to 98.6%, but increase again extraction time to recovery rate shadowRing little.
To XAD-7, XAD-2, NKA-9, AB-80 and the absorption property of D151 resin to anthocyaninEvaluate with desorption performance, result shows, XAD-7 has higher absorption property, is secondly XAD-2,And the effect of these three kinds of resins of NKA-9, AB-80 and D151 is all undesirable. Utilize 60% ethanol to enterRow is resolved, XAD-7, and AB-8 has higher desorption efficiency, but the latter's adsorbance is lower.
Solvent has impact to the elute effect of XAD-7 large pore resin absorption column: to ethanolic solution, withThe increase of the volume fraction of alcohol in ethanolic solution, in resin, anthocyanin desorption efficiency is in rising trend, and works asWhen volume fraction reaches 60%, reach desorb largely (desorption efficiency is 88%). Washing of methanol solutionSimilarly, the present invention adopts the ethanolic solution of low toxicity as eluent to de-effect.
As preferably, the frequency of ultrasonic extraction is 59kHz, and power is 500W.
As preferably, in step 1, remove ethanol and be specially: get extract through centrifugal, rotary evaporation in vacuo.
As preferably, the volume ratio of ethyl acetate and the first extract is 1:1.
As preferably, in step 2, remove ethyl acetate for water intaking layer rotary evaporation in vacuo.
As preferably, in step 2, the number of times of extraction is 3 times.
As preferably, the specification of XAD-7 macroporous resin column is 1.6cm × 40cm.
As preferably, the amount of distilled water is 5 times of column volumes.
As preferably, dry being specially in step 3: the first eluent after rotary evaporation, vacuum refrigerationDry 24h; Wherein, the temperature of rotary evaporation is no more than 40 DEG C.
As preferably, step 3 is specially: after the second extract loading and XAD-7 macroporous resin column, protectHold 1h, after the distilled water flushing with 5 times of column volumes, in post, add ethanolic solution to keep wash-out after 1h,Obtain the first eluent, rotary evaporation, vacuum refrigeration 24h.
As preferably, the specification of SephadexLH-20 gel column is 1.6cm × 60cm.
As preferably, in step 4, the flow velocity of wash-out is 1 drop/sec.
As preferably, in step 4, be dissolved between loading and also comprise with 0.45 μ m filtering with microporous membraneStep.
As preferably, dry being specially in step 4: the second eluent after rotary evaporation, vacuum refrigerationDry 24h; Wherein, the temperature of rotary evaporation is no more than 40 DEG C.
The present invention also provide make with preparation method provided by the invention 3,5-disaccharides anthocyanin.
As preferably, make with preparation method provided by the invention 3,5-disaccharides anthocyanin is that diformazan is spent kingfisherElement-3, the two glucosides of 5-O-.
The present invention also provide prepared by the method that the invention provides 3,5-disaccharides anthocyanin prepare food orApplication in medicine.
Provided by the invention 3, the preparation method of 5-disaccharides anthocyanin, comprise ultrasonic assisted extraction, extraction,The step of purifying. With ultrasonic assisted extraction reduced extractant use, shorten extraction time, improvedExtraction efficiency. The method that adopts gel column to combine with macroreticular resin, removes impurity, separates anthocyanin listBody, thus the use of preparation or half preparative high-performance liquid chromatographic avoided, simplify operation; And, solidifyingGlue post and macroreticular resin filler can Reusabilities, can economize on resources. Experimental results show that: ethanolic solution and firstIn alcoholic solution, pure volume fraction, the temperature and time of ultrasonic extraction are the key factors that affects extraction effect,Change any in these conditions and all can significantly reduce extraction efficiency. And in the purge process of step 2～4,The kind of macroporous absorbent resin, elution requirement are the key factors that improves purification effect. Employing the present invention carryThe method of confession is extracted every g mountain Grape Skin dry powder can obtain 7.36mg malvidin-3, the two glucose of 5-O-Glycosides, accounts for 98.7% of extracted anthocyanin, in addition, also comprises 0.324% delphinidin-3, the two grapes of 5-O-Glucosides and 0.841% methyl delphinidin 3, the two glucosides of 5-O-. Testing process has no other material peaks,Show to the invention provides 3 of method extraction, 5-disaccharides anthocyanin purity approaches 100%.
Brief description of the drawings
Fig. 1 shows fixing concentration of alcohol (60%), and ultrasonic treatment temperature and extractant volume extract anthocyaninThe impact of effect;
Fig. 2 shows fixing ultrasonic treatment temperature (50 DEG C), and concentration of alcohol and extractant volume affect anthocyanin and carryGet the impact of effect;
Fig. 3 shows concentration of alcohol and the reciprocal effect of extractant volume to anthocyanin content;
Fig. 4 shows HPLC testing result; Wherein, Fig. 4-a shows crude extract prepared by the embodiment of the present invention 3Chromatogram; Fig. 4-b show prepared by the embodiment of the present invention 43, the chromatogram of 5-disaccharides anthocyanin.
Detailed description of the invention
The preparation method who the invention provides a kind of 3,5-disaccharides anthocyanin, those skilled in the art can borrowMirror is content herein, suitably improves technological parameter and realizes. Special needs to be pointed out is all similar replacementsApparent to those skilled in the art with change, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, and related personnel obviously can be notDepart from content of the present invention, spirit and scope, methods and applications are herein changed or are suitably changed withCombination, realizes and applies the technology of the present invention.
The instrument that the present invention adopts is all common commercially available product, all can buy in market.
Wherein, mountain grape maturity fruit sample is taken from village experiment station in China Agricultural University for 2011.
The preparation of mountain Grape Skin powder is specially: under room temperature, pericarp is peeled off and be not with pulp, in liquid nitrogen middling speedFreeze, beat powder through agitator, under-40 DEG C of conditions, preserve. The fresh hill grape skin of every g is approximately prepared 0.3gMountain Grape Skin dry powder.
Below in conjunction with embodiment, further set forth the present invention:
The ultrasonic extraction conditions screening of anthocyanin in embodiment 1 mountain Grape Skin
Study respectively extract kind, volume fraction, solid-liquid ratio, ultrasonic extraction conditions in the Grape Skin of mountainThe impact of total anthocyanin extracted amount. The quantitative analysis of anthocyanin content adopts high performance liquid chromatography.
1, extract solvent species and the impact of volume fraction on the total anthocyanin extracted amount of mountain Grape Skin
Accurately take 0.5g mountain Grape Skin powder, add 2% formic acid methyl alcohol molten by liquid ratio 10:1 (mL/g)Liquid or 2% formic acid ethanolic solution, 50 DEG C of sonic oscillation temperature, ultrasonic extraction 20min. Extract solvent volumeMark is respectively 40%, 50%, 60%, 70%, 80%, 90%, 100%. Weight is established in each group experiment three timesMultiple, with anthocyanin extracted amount (mg/g, dry powder), for investigating index, solvent species and volume integral are extracted in researchSeveral impacts on total anthocyanin recovery rate in the Grape Skin powder of mountain, choose the suitableeest solvent species and volume fraction,Result is as shown in table 1:
Table 1 extracts reagent type and the impact of volume fraction on the total anthocyanin extracted amount of mountain Grape Skin
Note: the different letter representation significant differences of same column, horizontal p < 0.05 of otherness
Result shows, the anthocyanin content obtaining by acidic ethanol and acidic methanol extraction with aqueous solution is suitable,In this and anthocyanin, contain multiple phenolic hydroxyl groups, belong to water colo(u)r, be soluble in methyl alcohol, ethanol isopolarity is moltenAgent is relevant. Along with the increase of alcohol volume fraction, anthocyanin extracted amount increases, and exists but work as alcohol volume fractionAfter 60% time, anthocyanin content increasing degree tends towards stability. This point may with the penetrating power of solution graduallyIncrease and be correlated with, toxic while considering methyl alcohol as extractant, be unsuitable for extracting food coloring, Yi JiyouCan increase production cost and later stage thickening efficiency in higher volume fraction of ethanol, therefore select 60% acidityEthanolic solution is comparatively desirable.
2, the impact of formic acid concn on the total anthocyanin extracted amount of mountain Grape Skin
Fixing volumes of aqueous ethanol mark is 60%, under the condition of liquid ratio 10:1 (mL/g), in 50 DEG CUnder condition, extract total anthocyanin 20min of mountain Grape Skin powder, change formic acid volume fraction and be 0,2%, 4%,6%, 8%, 10%, three repetitions are established in each group experiment, use pH differential method to record the quality of total anthocyaninConcentration, determines optimum extraction temperature.
The impact of table 2 formic acid concn on the total anthocyanin extracted amount of mountain Grape Skin
| Volume fraction | 0 | 2％ | 4％ | 6％ | 8％ | 10％ |
| Anthocyanin content (mg/g) | 1.69b | 1.84a | 1.85a | 1.85a | 1.87a | 1.90a |
Note: the different letter representation significant differences of same column, horizontal p < 0.05 of otherness
Result shows, adding of formic acid has obvious impact to extraction effect, and the acidification of solution is describedAlso can improve the recovery rate of anthocyanin, but under strong acid condition, long-time heating is extracted, and easily occursThe partial hydrolysis of anthocyanin, is therefore fixed as 2% by formic acid volume fraction in this experiment.
3, the impact of liquid ratio on the total anthocyanin extracted amount of mountain Grape Skin
The mountain Grape Skin powder 0.5g that gets pulverizing, extraction conditions is: employing 2% formic acid 60% volume fractionEthanolic solution extracts 20min, sonic oscillation temperature 50 C, the body of ethanolic solution and mountain Grape Skin powderLong-pending mass ratio is respectively 5:1,10:1,15:1,20:1,25:1,30:1 (mL/g), and each group experiment establishes threeInferior repetition, measures total anthocyanin mass concentration, determines optimum extraction solvent volume.
The impact of table 3 liquid ratio on the total anthocyanin extracted amount of mountain Grape Skin
| Volume mass is than (mL/g) | 5:1 | 10:1 | 15:1 | 20:1 | 25:1 | 30:1 |
| Anthocyanin content (mg/g) | 1.30cd | 1.96a | 1.56ab | 1.52ab | 0.96cd | 0.62d |
Note: the different letter representation significant differences of same column, horizontal p < 0.05 of otherness
Result shows, recovery rate increases with ethanolic solution and the increase of the volume mass ratio of mountain Grape Skin powder,No longer rise when being increased to after 10:1, therefore, select liquid ratio 10:1 to be advisable.
4, the impact of extraction time on the total anthocyanin extracted amount of mountain Grape Skin
By mountain Grape Skin powder, taking mass volume ratio as 1:10, (g/mL) mixes with ethanolic solution, wherein ethanolIn solution, the volume fraction of each component is: ethanol 60%, formic acid 2%, water 38%. 53.5 DEG C of ultrasonic carryingGet 20min. Repeat to extract 8 times. After each extraction, measure the content of anthocyanin in extract, determineGood extraction time.
The impact of table 4 extraction time on the total anthocyanin recovery rate of mountain Grape Skin
Note: the different letter representation significant differences of same column, horizontal p < 0.05 of otherness
Result shows: along with the increase of extraction time, anthocyanin recovery rate increases, while extraction for the third time,Anthocyanin extraction rate reached to 98.6%, has significantly improved recovery rate, then increases extraction time recovery rate is affectedNot quite, in practical operation, for saving operating time and experimentation cost, adopt three grades of extractions.
5, the impact of ultrasonic time on the total anthocyanin extracted amount of mountain Grape Skin
Extract is 2% formic acid 60% ethanolic solution, under the condition of liquid ratio 10:1 (mL/g), inUnder 50 DEG C of conditions, extract the total anthocyanin in Grape Skin, extraction time is respectively 10min, 20min, 30Min, 40min, 50min, 60min, three repetitions are established in each group experiment, and ultrasound condition is fixed as ultrasonicFrequency 59KHz, power 500W, duty 100%. Use pH differential method to record total anthocyaninMass concentration, determine the optimum extraction time.
The impact of table 5 ultrasonic time on the total anthocyanin extracted amount of mountain Grape Skin
| Ultrasonic time (min) | 10 | 20 | 30 | 40 | 50 |
| Anthocyanin content (mg/g) | 1.55ab | 1.88a | 1.71ab | 1.61ab | 1.50b |
Note: the different letter representation significant differences of same column, horizontal p < 0.05 of otherness
Result shows: in the time that extraction time is 20min, extract comparatively abundant, with extraction time extend,Extracted amount declines slightly, may be because long-time high temperature extracts, and causes anthocyanin decomposes. ThereforeTo extraction time be fixed on 20min.
6, the impact of ultrasonic temperature on the total anthocyanin extracted amount of mountain Grape Skin
Be 2% formic acid 60% ethanolic solution at extract, under the condition of liquid ratio 10:1 (mL/g), inAt 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, extract the total anthocyanin 20min in Grape Skin,Three repetitions are established in each group experiment, and ultrasound condition is fixed as supersonic frequency 59KHz, power 500W, workState 100%. Use pH differential method to record the mass concentration of total anthocyanin, determine optimum extraction temperature.
The impact of the ultrasonic temperature of table 6 on the total anthocyanin extracted amount of mountain Grape Skin
| Ultrasonic temperature (DEG C) | 20 | 30 | 40 | 50 | 60 | 70 |
| Anthocyanin content (mg/g) | 1.35b | 1.51b | 1.72a | 1.80a | 1.87a | 1.48b |
Note: the different letter representation significant differences of same column, horizontal p < 0.05 of otherness
Result shows: in the scope of 70 DEG C, with extract the rising of temperature, total anthocyanin extracted amount is correspondingIncrease, in the time that temperature reaches 40 DEG C, extracted amount reaches peak, and and 30 DEG C, 70 DEG C locate significant difference(P < 0.05), not remarkable with 50 DEG C, 60 DEG C differences. Therefore select 40 DEG C for preference temperature. This is mainBe because anthocyanin material heat resistance is poor, long-time high temperature will cause the degraded of anthocyanin.
7, optimum extraction condition is determined in response surface experiment
Selective extraction agent volume, extraction temperature and 3 of volume fraction of ethanols affect anthocyanin extraction effectLarger factor is carried out 3 factor 5 hydraulic tests, and experimental design and result are as shown in table 7:
Table 7 center combination design experiment result
As shown in Table 7, select Central Composite model, do the 3 factor 5 levels responses of totally 20 testing sitesSurface analysis test. Wherein tested number 1-14 is factorial test, and tested number 15-20 is center test, usesEstimate test error. Utilize SASRSREG program, adopt regression equation Y＝α0+α1X1+α2X2+α3X3+α11X1 2+α22X2 2+α33X3 2+α12X1X2+α13X1X3+α23X2X3, his-and-hers watches 7Data are processed, and regression coefficient and the results of analysis of variance are in table 8.
Table 8 response surface regression coefficient value
| Coefficient | Estimation coefficient | Conspicuousness |
| α0 | -1.05872 | ** |
| α1 | 0.25827 | ** |
| α2 | 0.03879 | ** |
| α3 | 0.04850 | ** |
| α11 | -0.02530 | ** |
| α12 | -0.00075 | NS |
| α13 | 0.00120 | NS |
| α22 | -0.00033 | *** |
| α23 | 0.00002 | NS |
| α33 | -0.00047 | *** |
| Model | *** | |
| Lose and intend | NS | |
| R2 | 0.9055 |
Note: * p<0.05, * * p<0.01, * * * p<0.001, NSp>0.1
In regression equation, the conspicuousness of each variable on index (response) impact, is checked to judge by F,Probability P value is less, and the significance degree of relevant variable is higher. As can be seen from Table 8, model returns effectFruit is extremely remarkable, and model loses and intend significantly (P > 0.1) and coefficient of determination R2Be 0.9055, this model is describedFitting degree is good. Linear relationship between independent variable and response is remarkable, and the theory that can be used for experiment is pre-Survey. Experimental result shows extractant volume (X1), ultrasonic temperature (X2), volume fraction of ethanol (X3)Once all result of the test tool is had a significant impact to (P < 0.01) with quadratic term, and the impact of mutual is notSignificantly. By rejecting inapparent of result of the test impact, the model equation after being optimized as follows:
Y＝0.25827X1+0.038790X2+0.048498X3-0.025296X1 2-0.00033X2 2-0.00047X3 2-1.05872
Fig. 1～3 are the intuitive analysis figure of each test factor to mountain Grape Skin anthocyanin extraction effect, can be directly perceivedFind out the reciprocal effect between each factor:
What Fig. 1 reflected is to fix after concentration of alcohol (60%), and ultrasonic treatment temperature and extractant volume are to flowerThe impact of look glycosides extraction effect. From figure, data can be found out in the time of extractant constancy of volume, ultrasonic processingTemperature plays " quadratic effect " to result of the test, along with the increase of ultrasonic treatment temperature, and anthocyanin contentObviously increase, but in the time exceeding 55 DEG C of left and right, anthocyanin content starts sharply to decline; Get ultrasonic processingTemperature is fixed value, as 50 DEG C, is increased to 5.7mL with extractant volume, and anthocyanin content obviously raisesTo 2.14mg/g, the variation of anthocyanin content afterwards tends towards stability, no longer with the increase of extractant volumeVariation.
When Fig. 2 is fixing ultrasonic treatment temperature (50 DEG C), concentration of alcohol and extractant volume affect patternThe schematic diagram of glycosides extraction effect. In the time extracting constancy of volume, along with the increase of concentration of alcohol, anthocyanin containsAmount obviously increases, and in the time that concentration of alcohol is 60%, anthocyanin content reaches peak, and anthocyanin contains afterwardsAmount obviously declines; Fixing concentration of alcohol, impact and Fig. 2-7-A of extractant volume on anthocyanin extraction effectSimilar, along with the increase of extractant volume, anthocyanin content be first increase after substantially constant trend.
Fig. 3 shows concentration of alcohol and the reciprocal effect of extractant volume to anthocyanin content, presents similar " twoInferior effect " trend.
To sum up, use DesignExpert software to analyze regression equation, draw best anthocyaninExtraction process condition is: by mountain Grape Skin powder and ethanolic solution taking mass volume ratio as 1:10 (g/mL) mixedClose, wherein in ethanolic solution, the volume fraction of each component is: ethanol 60%, formic acid 2%, water 38%. 53.5 DEG CUltrasonic extraction 20min. Repeat to extract three times. Wherein ultrasound condition is fixed as supersonic frequency 59KHz, meritRate 500W, duty 100%. After extraction, sample liquid is at 8000r/min, centrifugal under 4 DEG C of conditions10min, and get clear liquid after 0.45 μ m miillpore filter vacuum filtration, under lower than 40 DEG C of conditions, carry out verySky revolves and boils off except ethanol, obtains anthocyanin and extracts concentrate.
Embodiment 2 ethyl acetate liquid-liquid extractions
The first extract is mixed in isopyknic ethyl acetate, carries out liquid-liquid extraction, collect lower aqueous layer,In triplicate, combining water layer, and carry out vacuum under lower than 40 DEG C of conditions and revolve and boil off except ethyl acetate,Obtain the second extract.
Adopt ethyl acetate extraction can tentatively remove in solution other low pole classes such as flavanone materialMaterial.
The purification condition screening of embodiment 3 macroreticular resins
1, the screening of macroporous absorbent resin
Get XAD-7, XAD-2, NKA-9, AB-80 and D151 resin and carry out after pretreatment, each standardReally measure 5mL, blot resin surface moisture with filter paper, be placed in 100mL tool plug ground triangular flask,Add the anthocyanin solution 5mL of certain mass concentration, under room temperature in isothermal vibration device with 130r/minSpeed carry out lucifuge vibration absorption 1h, fully, after absorbing and filtering, measure the light absorption value of anthocyanin in filtrate,Calculate the saturated extent of adsorption Q of Grape Skin anthocyanine(mg/L); By the resin after filtering with 5 times of column volumesDistilled water flushing, adds the ethanolic solution of 60% volume fraction of 10mL2% formic acid acidifying to carry out 1h to itStatic desorption experiment. Measure the light absorption value of resolving anthocyanin in solution, calculate thus the large of different modelThe desorption quantity Q of hole resin to Grape Skin anthocyanind(mg/L). With the saturated extent of adsorption of macroporous absorbent resin andDesorption quantity and desorption efficiency D (%) are index, investigate the absorption property of various macroreticular resins, filter out bestPolymeric adsorbent.
Computing formula is:
In formula: C0For the initial mass concentration (mg/L) of anthocyanin; C1For quality after anthocyanin resin adsorptionConcentration (mg/L); C2For mass concentration (mg/L) after anthocyanin resin desorption; V1For anthocyanin extractantVolume (mL); W is resin humid volume (mL); VdFor the volume (mL) of stripping liquid.
Result of calculation is as shown in table 9:
Table 9 resinous type and physical arrangement parameter
Note: the different letter representation significant differences of same column, horizontal p < 0.05 of otherness
Result shows, XAD-7 has higher absorption property, is secondly XAD-2, and NKA-9,The effect of these three kinds of resins of AB-80 and D151 is all undesirable. Utilize 60% ethanol to resolve,XAD-7, AB-8 has higher desorption efficiency, but the latter's adsorbance is lower.
2, the screening of eluting solvent
Conventional eluant, eluent has methyl alcohol, ethanol, simultaneously in order to make anthocyanin remain more stable in solutionPattern melt cation state, need to carry out certain acidifying to eluant, eluent, common acidulant have hydrochloric acid,Formic acid etc. In order to determine the optimum solvent of XAD-7 macroreticular resin wash-out, get 5mL saturated adsorption patternIn the XAD-7 resin of glycosides, change eluent (methanol solution or ethanolic solution, volume is all 10mL)Volume fraction and one of two kinds of variablees of volume fraction of acidified solvent (formic acid), under room temperature, shake at constant temperatureThe speed with 130r/min in device of swinging is shaken desorb 1h, measures pattern in the solution of desorption experiment front and backThe mass concentration of glycosides, calculates resolution factor.
Wherein, when fixed acid agent formic acid volume fraction is 2%, eluting solvent methyl alcohol and volume fraction of ethanolImpact on anthocyanin material desorption effect is as shown in table 10:
Table 10 variety classes, different volumes mark the impact of eluent on desorption efficiency
Note: the different letter representation significant differences of same column, horizontal p < 0.05 of otherness
Result shows, for methyl alcohol, ethanol, it imitates anthocyanin wash-out under different volumes markThe impact similar (P > 0.05) of fruit, and along with the increase of volume fraction separately, anthocyanin desorption efficiency in resinIn rising trend, and in the time that volume fraction reaches 60%, (desorption efficiency is respectively to reach desorb largelyBe 85%, 88%), along with the continuation of volume fraction raises, desorption effect does not have significant change afterwards. ExamineConsider the characteristic biodegradable, nontoxic to ethanol and the security of gained anthocyanin, in fixing firstUnder the acid volume fraction prerequisite that is 2%, select 60% ethanol as more excellent eluant, eluent.
After fixing volume fraction of ethanol (60%), formic acid acidulant volume fraction is to anthocyanin elute effectAffect as shown in table 11:
The impact of the different formic acid volume fractions of table 11 on resolution factor
Note: the different letter representation significant differences of same column, horizontal p < 0.05 of otherness
Result shows, by add formic acid in eluant, eluent, can significantly improve XAD-7 resin to anthocyaninElute effect, the increase of formic acid volume fraction, at volume fraction 2%-8%, between there is no conspicuousnessDifference (P > 0.05), be 2% so finally select formic acid volume fraction. The eluant, eluent of this test is finally trueBe decided to be 60% ethanolic solution of 2% formic acid acidifying.
3, adopt screening gained condition purifying to the second extract
The ethanolic solution of 60% (volume fraction) of the acidifying of employing 2% (volume fraction) formic acid is as macroporeThe eluent of resin XAD-7, before macroreticular resin XAD-7 uses, adopts the eluent of 1 times of column volumeCarry out balance.
Get the second extract obtaining in the embodiment of the present invention 2 and be splined on XAD-7 macroporous absorbent resin, extremely2/3rds of column volume is colored. Rinse to remove the second extraction with the distilled water of 5 times of column volumesThe impurity such as sugar in liquid, organic acid, protein, then adopt 60% ethanol water of 2% formic acid acidifyingCarry out wash-out, collect colored the first eluent. After the condition backspin lower than 40 DEG C steams, vacuum is coldThe dry 24h of freeze-drying becomes powder to be crude extract.
Before and after purifying, the look valency measurement result of anthocyanin is as shown in table 12:
The look valency measurement result of table 12 anthocyanin product
| State | Before purifying | After purifying |
| Look valency | 40.6 | 168.7 |
Result shows, after XAD-7 purifying, the look valency of anthocyanin has improved more than 3 times before than purifying, purifyingAfter anthocyanin powder be aubergine.
Embodiment 4SephadexLH-20 gel column purifying
1, the pretreatment of SephadexLH-20 gel column and dress column method
Get the dry glue of 27gSephadexLH-20, add the abundant swelling 3h of 40mL water, swelling process shouldAvoid undue agitation and use magnetic stirring apparatus. Gel after swelling is placed in to vacuum desiccator to be vacuumized1h, removes the bubble in colloid, after stirring, utilizes glass bar drainage, continuously introduced layerAnalyse in post, sedimentation is spent the night. For preventing cylinder hollow out or generating bubble, can be by ultrasonic eluant, eluent processing 20Min, and reduce eluant, eluent gradient span as far as possible.
2, with SephadexLH-20 gel column purification of crude extract
SephadexLH-20 gel column should be first before using with 2% (volume fraction) formic acid of 2 times of column volumes20% (volume fraction) methanol solution of acidifying carries out balance.
Afterwards the crude extract of preparation in example 3 is dissolved in to 20% (body of 2% (volume fraction) formic acid acidifyingIntegration number) methanol solution is to saturated, after 0.45 μ m filtering with microporous membrane, loading 2mL to SephadexLH-20 gel column (specification of post is 1.6cm × 60cm), with 20% methanol solution (2% formic acid acidifying)Carry out wash-out separation, collect the second eluent, after the condition backspin lower than 40 DEG C steams, vacuum refrigeration is dryDry 24h becomes powder to be 3,5-disaccharides anthocyanin.
Embodiment 5 with the invention provides prepared by method 3,5-disaccharides anthocyanin Quality Identification
Taking 20% (volume fraction) methanol solution of 2% (volume fraction) formic acid acidifying as solvent, dissolveEmbodiment 4 prepare 3,5-disaccharides anthocyanin, and dissolve crude extract in contrast. Adopt HPLC-MSProducts therefrom is carried out to qualitative and quantitative analysis.
HPLC-MS condition is: Kromasil100-5-C18 chromatographic column (250mm × 4.6mmI.D.), streamMoving phase A consists of water: formic acid=90:10 (v/v), Mobile phase B consist of water: methyl alcohol: formic acid=40:50:10 (v/v/v). Elution program: 0-4min, 6-15%B; 4-13min, 15-25%B; 13-20 min，25-50％B；20-35min，50-80％B；35-40min，80-100％B；40-45min，100-6%B; Flow velocity is 1.0mL/min; Column temperature is 50 DEG C; Sample size is 30 μ L, and chromatogram is at 525nmUnder wavelength, detect and record, wherein the corresponding purity of anthocyanin monomer is according to peak area institute in chromatogramThe ratio accounting for is calculated.
Adopt malvidin-3-O-glucoside to make external standard, taking anthocyanin concentration as abscissa, with HPLCDetected peaks area is ordinate, sets up between 5-500mg/L, the standard song of 9 levels, three repetitionsLine, regression equation is Y=46.01304X-6.99129, coefficient correlation reaches 0.9998. Be used for the invention providesMethod makes the quantitative analysis of 3,5-disaccharides anthocyanin.
Mass spectrum adopts electric spray ion source (ESI), positive ion mode, ion scan scope: 100-1500m/z;Atomizer pressure: 30psi; Dry gas flow velocity: 12L/min; Dry gas temperature: 300 DEG C. Sample processDirect injection analysis after 0.45 μ m membrane filtration, every sample in triplicate. Testing result is with reference to HeF, LiangN,MuL,etal.AnthocyaninsandtheirvariationinredwinesI.MonomericAnthocyaninsandtheircolorexpression.Molecules, 2012,17 (2): 1571-1601. providesIn grape and grape wine, anthocyanin HPLC-DAD-MS fingerprint spectrum library carries out qualitative.
HPLC detects collection of illustrative plates as shown in Figure 4. Wherein, Fig. 4-a shows prepared by the embodiment of the present invention 3 slightly carryingThe chromatogram of thing; Fig. 4-b show prepared by the embodiment of the present invention 43, the chromatogram of 5-disaccharides anthocyanin.Can find out, SephadexLH-20 gel column purifying has improved peak 3 materials greatly at total anthocyanin materialIn ratio, reduced the content of impurity. And the chromatogram shown in Fig. 4-b shows, detects altogether threePlant anthocyanin material. These three kinds of materials are done to further Mass Spectrometer Method, and testing result is as shown in table 13:
The HPLC-MS feature of table 13 anthocyanin after SephadexLH-20 gel column separates
Result shows: in Fig. 4-b, No. 3 peak materials are the anthocyanin component separating at SephadexLH-20In be topmost component, account for 98.7%, molecular ion is 655m/z, fragment ion is493m/z ([M-C6H10O5]+), 331m/z ([M-C6H10O5-C6H10O5]+). In this process, loseLosing two glucose molecules, is malvidin-3 by this material preliminary judgement, the two glucosides of 5-O-(Malvidin-3,5-O-diglucoside). Quantitative result demonstration, the content of this material is 7.36mg/g (PortugalGrape skin dry powder).
In Fig. 4-b, No. 1 and No. 2 peak materials are respectively delphinidin-3, the two glucosides of 5-O-(Delphinidin-3,5-O-diglucoside) and methyl delphinidin-3, the two glucosides of 5-O-(Petunidin-3,5-O-diglucoside) loses respectively two glucose molecules, obtains m/z303,317Molecular fragment, account for respectively 0.324% and 0.841%.
In chromatogram testing process, except peak 1～3, other material peaks do not detected, show that the present invention carriesThe method of confession prepare 3,5-disaccharides anthocyanin purity is higher, approaches 100%.
Below be only the preferred embodiment of the present invention, it should be pointed out that the common skill for the artArt personnel, under the premise without departing from the principles of the invention, can also make some improvements and modifications,These improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. one kind 3, the preparation method of 5-disaccharides anthocyanin, is characterized in that, comprises the following steps:
Step 1: with the ultrasonic extraction of ethanolic solution mountain Grape Skin powder, extract is removed ethanol, makes first and carriesGet liquid;
Step 2: extract described the first extract with ethyl acetate, water intaking layer, removes ethyl acetate and make theTwo extracts;
Step 3: described the second extract is splined on to XAD-7 macroporous resin column, after distilled water flushing,With ethanolic solution wash-out, obtain the first eluent, drying, makes crude extract;
Step 4: described crude extract, with dissolve with methanol solution, is splined on to SephadexLH-20 gel column,With methanol solution wash-out, obtain the second eluent, drying, to obtain final product;
Wherein, in described ethanolic solution, the volume fraction of each component is: ethanol 60%, formic acid 2%, water38％；
Described 3,5-disaccharides anthocyanin is malvidin-3, the two glucosides of 5-O-, delphinidin-3,5-O-Two glucosides and methyl delphinidin 3, the two glucosides of 5-O-;
In described methanol solution, the volume fraction of each component is: methyl alcohol 20%, formic acid 2%, water 78%;
The mass volume ratio of described mountain Grape Skin powder and described ethanolic solution is: 1g:10mL;
The temperature of described ultrasonic extraction is 53.5 DEG C, and the time is 20min, and number of times is 3 times.
2. preparation method according to claim 1, is characterized in that, the frequency of described ultrasonic extractionFor 59kHz, power is 500W.
3. preparation method according to claim 1, is characterized in that, described ethyl acetate with described inThe volume ratio of the first extract is 1:1.
4. preparation method according to claim 1, is characterized in that, described XAD-7 macropore treeThe specification of fat post is 1.6cm × 40cm.
5. preparation method according to claim 1, is characterized in that, described SephadexLH-20The specification of gel column is 1.6cm × 60cm.
6. preparation method according to claim 1, is characterized in that, dry tool described in step 3Body is: the first eluent after rotary evaporation, vacuum freeze drying 24h; Wherein, the temperature of rotary evaporationDegree is no more than 40 DEG C.
7. preparation method according to claim 1, is characterized in that, wash-out described in step 4Flow velocity is 1 drop/sec.
8. preparation method according to claim 1, is characterized in that, dry tool described in step 4Body is: the second eluent after rotary evaporation, vacuum freeze drying 24h; Wherein, the temperature of rotary evaporationDegree is no more than 40 DEG C.
9. make with preparation method described in claim 1～8 any one 3,5-disaccharides anthocyanin.
10. make with preparation method described in claim 1～8 any one 3,5-disaccharides anthocyanin preparationApplication in food or medicine.
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| CN112175028B (en) * | 2020-09-14 | 2021-09-21 | 浙江大学 | Method for separating and preparing delphinidin-3-O- (6-O-p-coumaroyl) glucoside |
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